Liposomes can serve as vesicles to deliver a wide range of encapsulated and/or membrane-incorporated therapeutic or diagnostic entities. Liposomes are usually characterized as nano-scale vesicles consisting of an interior core separated from the outer environment by a membrane of one or more bilayers. The bilayer membranescan be formed by amphiphilic molecules e.g. synthetic or natural lipids that comprise a hydrophobic and a hydrophilic domain [Lasic, Trends Biotechnol., 16: 307-321, 1998]. Bilayer membranes can also be formed by amphiphilic polymers constituting particles (e.g. polymersomes and polymerparticles).
Liposomes can serve as carriers of an entity such as, without limitation, a chemical compound, or a radionuclide, that is capable of having a useful property or provide a useful activity. For this purpose, the liposomes are prepared to contain the desired entity in a liposome-incorporated form. The liposome incorporated entity can be associated with the exterior surface of the liposome membrane, located in the interior core of the liposome or within the bilayer of the liposome. Methods for the incorporation of radionuclides into liposomes are e.g. surface labeling after liposome preparation [Phillips, Adv Drug Deliv Rev., 37: 13-32, 1999], label incorporation into the lipid bilayer of preformed liposomes [Morgan et al., J Med. Microbiol., 14: 213-217, 1981], surface labeling of preformed liposomes by incorporating lipid chelator conjugate during preparation [Goto et al., Chem harm Bull. (Tokyo), 37: 1351-1354, 1989; Seo et al., Bioconjucate Chem., 19: 2577-2584, 2008], and aqueous phase loading of preformed liposome [Hwang et al., Biochim Biophys Acta., 716: 101-109, 1982; Phillips et al., Int J Rad Appl Instrum B, 19: 539-547, 1992; Gabizon et al., J Liposome Res., 1: 123-125, 1988; Henriksen et al., Nucl Med. Bio., 31: 441-449, 2004]. The incorporation of entities into liposomes by the aqueous phase loading of preformed liposome is also referred to as “loading” and thereby “encapsulating” or “entrapping” the entities.
Encapsulating entities into the interior of liposomes through aqueous phase loading seems to provide the greatest in vivo stability, because of the protected location of the entity inside the liposome. The purpose of encapsulating an entity into a liposome is often to protect the entity from the destructive environment and rapid excretion in vivo. The entrapment of the entity provides the opportunity for the encapsulated entity to apply the activity of the entity mostly at the site or in the environment where such activity is advantageous but less so at other sites where the activity may be useless or undesirable. It is known that liposomes having PEG chains attached to the outer surface have prolonged circulation time in the blood stream. These liposome compositions can effectively evade the immune system, which would otherwise attack the liposomes soon after injection causing fast clearance or rupture of the liposome and premature release of the agent entrapped inside. By increasing the blood circulation time, the agent entrapped in the liposome stays within the liposome until it reaches the target tissue. This phenomenon is referred to as passive targeting delivery, where an accumulation of long-circulating nanoparticles in tumor areas or inflammatory sites is due to leaky vasculature and lack of an effective lymphatic drainage system in these areas. For example, a radio-diagnostic entity entrapped within a long-circulating liposome can be delivered by passive targeting to a diseased site within a subject to facilitate a diagnosis thereof. Active- or ligand targeting delivery systems is referred to liposome compositions with ligands attached on the surface targeted against cell surface antigens or receptors [Allen, Science, 303: 1818-1822, 2004]. Combining the properties of targeted and long-circulating liposomes in one preparation comprising a radionuclide encapsulated liposome composition would significantly enhance the specificity and intensity of radioactivity localization in the target site e.g. a tumor. Ideally, such liposome compositions can be prepared to include the desired entity, e.g. a chemical compound or radionuclide, (i) with a high loading efficiency, i.e., high percentage of encapsulated entity relative to the total amount of the entity used in the encapsulation process, and (ii) in a stable form, i.e., with minimal release (i.e. leakage) of the encapsulated entity upon storage or generally before the liposome reaches the site or the environment where the liposome entrapped entity is expected to apply its intended activity.
Entrapment of radionuclides into nanoparticles such as liposomes can be obtained through use of chemical compounds called ionophores capable of transporting metal ions across lipid membranes. Upon crossing the membrane barrier the radionuclide then binds preferably to a chelator, encapsulated in the interior of the liposome composition, due to its stronger affinity thereto, allowing the release of free ionophore, and the entrapment of the radionuclide in the liposome composition.
Copper isotopes are of great interest for use in diagnostic and/or therapeutic application. For diagnostic applications this relates to the positron-emitters 61Cu and 64Cu, which can be used in positron emission tomography (PET) diagnostic imaging. 64Cu is an interesting copper isotope possessing all decay modalities, and with a half-life of 12.7 h it is favorable for biological studies. A half-life of about 6-12 h appears to be ideal to allow for sufficient accumulation of liposome in inflammatory tissues or cancerous targets, yet providing enough background clearance to permit early identification of the target [Gabizon et al., Cancer Res., 50: 6371-6378, 1990]. Furthermore, 64Cu can be used as a model nuclide representing the chemical properties of all copper isotopes.
Ideal radioisotopes for therapeutic applications are those with low penetrating radiation, such as β-, α- and auger electron-emitters. Examples of such radioisotopes are 67Cu, 67Ga, 225Ac, 90Y, 177Lu and 119Sb. When the low energy emitting radioisotope in the form of a radiopharmaceutical reach the target site, the energy emitted is only deposited at the target site and nearby normal tissues are not irradiated. The energy of the emitted particles from the different radioisotopes and their ranges in tissues will vary, as well as their half-life, and the most appropriate radioisotope will be different depending on the application, the disease and the accessibility of the disease tissue.
Ideal radioisotopes for diagnostic applications are those with relatively short half-life, and those with high penetrating radiation to be detected by imaging techniques such as positron emission tomography (PET) and/or single photon emission computed tomography (SPECT). The half-life of the radionuclide must also be long enough to carry out the desired chemistry to synthesize the radiopharmaceutical and long enough to allow accumulation in the target tissue in the patient while allowing clearance through the non-target organs. The radionuclide, 64Cu, has proven to be a versatile isotope with respect to is applications in both imaging [Dehdashti et al., J Nucl Med. 38: 103P, 1997] and therapy [Anderson et al., J Nucl Med., 36: 2315-2325, 1998]. Radiopharmaceuticals and for example radiolabeled liposome compositions consisting of radionuclides, such as 61Cu (T½=3.33 h) and 64Cu (T½=12.7 h) can be utilized for imaging by the positron emission tomography (PET) technique, with the main advantages over single photon emission computed tomography (SPECT) being: a) employing annihilation coincidence detection (ACD) technique whereby only photons detected simultaneously (<10−9 sec) by a pair of scinitillators opposite each other are registered, instead of collimator, the sensitivity is markedly improved (×30-40) and the spatial resolution is enhanced by about a factor of two (<5 mm), since the detection field is (non-diverging) defined cylindrical volume and both the sensitivity and the resolution do not vary within the detection field [Kostarelos et al., J Liposome Res., 9: 429-460, 1999]; b) PET scanners provide all images in the unit of radioactivity concentrations (e.g. Bq/ml) after corrections for photon attenuation, scatters and randoms, thereby considering PET to be a more quantitative technique than SPECT [Seo, Curr. Radiopharm., 1: 17-21, 2008].
The patent applications WO/2001/060417, WO/2004/082627, WO/2004/082626 and US 20090081121, describe methods based on ionophoric loading of radionuclides into liposomes. Further, the disclosed radionuclides which are loaded into liposomes are heavy radionuclides and 11C, 18F, 76Br, 77Br, 89Zr, 67Ga, 111In, 177Lu, 90Y and 225Ac. From a diagnostic standpoint, these approaches are not useable for PET imaging applications, but only SPECT, because of the limited use of radionuclides.
Patent EP386 146 B1 describes a composition and method of use for liposome encapsulated compounds for neutron capture tumor therapy. However, these liposomes were loaded with stable elements (e.g. boron), that become radioactive only after activation.
In a theoretical study, Kostarelos et al., analyzed the therapeutic potential of liposomes labeled with one of the radionuclides 131I, 67Cu, 188Re or 211At, but chemical procedures for the preparation of the labeled liposomes were not suggested [Kostarelos et al., J Liposome Res, 9:407-424, 1999].
Only a few radiopharmaceuticals based on radioactive copper isotopes are discovered and available today. Examples are 60Cu-ATSM as hypoxia marker, and 64Cu-ATSM and 64Cu-PTSM, which are suggested as potential agents for tumor therapy. Further classes of substances are copper-labeled peptides and antibodies in which the radioactive copper is linked to the biomolecule via a bifunctional chelator. There are no liposome compositions loaded with copper available for use as radiopharmaceuticals.
Several research groups have measured the permeability of anions and cations through lipid bilayers without the use of ionophores.
It is known in the field that the low ion permeability of phospholipid bilayers such as liposome compositions [Paula et al., Biophys. J., 74:319-327, 1998; Hauser et al., Nature, 239:342-344, 1972; Ceh et al., J. Phys. Chem. B, 102:3036-3043, 1998; Mills et al., Biochim. Biophys. Acta, 1716:77-96, 2005; Papahadjopoulos et al., Biochim. Biophys. Acta, 266:561-583, 1971; Puskin, J. Membrane Biol, 35:39-55, 1977] leads to highly unfavorable loading kinetics for charged ion species. Therefore it is common practice to use an ionophore to increase trans-bilayer diffusion rates, and thereby improve or increase the loading of monovalent, divalent and trivalent cations into nanoparticles such as liposomes.
The patent application WO2006/043083 describes a method for loading of radionuclides, which involves ionophores and chelators. It is mentioned in the application that a chelator may be an ionophore.
The patent application WO03/041682 discloses liposomes enclosing biological agents. It is disclosed in the application that ion-gradients, ionophores, pH gradients and metal complexation procedures can be used for active loading of liposomes with biological agents. The application does not disclose a method for loading of nanoparticles with metal entities wherein an osmotic gradient is used to increase the loading efficiency or loading rate.
There is a need in the technical field of diagnostic applications to provide various liposome compositions that are useful for delivery of a variety of compounds, for example radio-diagnostic and imaging entities useful for PET.